Salt tolerance of plants is important to both agriculture and environmental protection. Today, one third of the land on earth is said to be dry land. Further, it is anticipated that the proportion of dry land will increase in the future, due to the progressive desertification of both cultivated land and green land. Considering the prediction that the world population in the year 2050 will be 1.5 times that of today and the serious problems of provisions arising as a result, development of cultivars that grow on land ill-fitted for cultivation, especially on dry land, as well as cultivation techniques for the same is a matter of great urgency. The problem with agriculture on dry land is salt accumulation. In a dry climate, evapotranspiration outstrips precipitation and continued irrigation on land where much is desired for drainage leads to plenty of salt accumulation, due to the deposition of salt on the surface by acceleration of rise in subterranean water level that bear salinity. Examples where cultivation becomes impossible as a result are known from the ancient past, represented by the end of Tigris-Euphrates civilization. The problem still arises today. Thus, innovation of agriculture, on dry land and on land where salt is accumulated, to enhance the salt tolerance of plants is of great importance (Toshiaki Tanno (1983) Kagaku to Seibutsu 21:439-445 “Salt tolerance of crops and mechanism of the same”; Yasutaka Uchiyama (1988) Kagaku to Seibutsu 26:650-659 “Agricultural use of salinenvironment”).
There are two kinds of stress related to salt stress against plants, namely stress by osmotic pressure and stress by ionicity. An osmotic pressure stress is a stress whose action is the same as the stress by dehydration. It results from high osmotic pressure, due to high salinity environment around the plant, which leads to a setback of water absorption of the plant and at the same time deprivation of water from the plant body. It is known that a mechanism exists in the plant to avoid the osmotic pressure stress. The core substances associated with this function are ions (such as K+, Na+, Cl−, organic acid, etc.) as well as substances called compatible solutes. The term “compatible solute” refers to substances such as sugar, proline (a kind of amino acid), and glycine betaine (a quaternary ammonium compound), and so on, which do not disturb the metabolic pathway or inhibit enzymatic action, even when accumulated at a high concentration in the cell. Plant cells accumulate these substances which, in turn, preserve the osmotic pressure balance to the external world (Manabu Ishitani, Keita Arakawa, and Tetsuko Takabe (1990) Chemical Regulation of Plants 25:149-162, “Molecular mechanism of salt tolerance in plants”).
Almost no development has been made regarding the mechanism of plants to avoid ionic stress. Absorption of excess Na+ by the plant cell leads to inhibition of intracellular enzyme reaction and finally to metabolic trouble (Toru Matoh (1997) Chemical Regulation of Plants 32:198-206, “Salt tolerance mechanism of the plant”). Therefore, it is necessary to eliminate the intracellulary accumulated Na+ from the cell or isolate it into intracellular organs, such as vacuoles. The Na+/H+ antiporter (sodium/proton antiporter) is assumed to play the central role in this process. The Na+/H+ antiporters of plant cells are thought to exist on both the cell membrane and the vacuolar membrane. They utilize the pH gradient formed between the biomembranes by the H+ pump (H+-ATPase and H+-PPase), an element that transports H+ as the energy to transport Na+ existing in the cytoplasm out of the cell or into the vacuole. Moreover, it is presumed that plants contacted with salt of high density, have to retain intercellular K+/Na+ ratio high enough, maintaining the osmotic pressure balance between the cell exterior and interior by accumulating Na+ in the vacuole through the Na+/H+ antiporter.
The Na+/H+ antiporters found existing on plasma membrane are well examined in animals, yeasts, bacteria and so on. On the plasma membrane of an animal cell, H+ is carried by the Na+/H+ antiporter, to maintain the balance of H+ in the cell, utilizing the Na+ concentration gradient between the membranes formed by Na+/K+-ATPase. Therefore, the antiporter is presumed to be deeply related with intracellular pH modulation, control of the cell volume, as well as Na+ transport through the plasma membrane (Orlowski, J. and Grinstein, S. (1997) J.Biol.Chem. 272:22373-22376; Aronson, P. S. (1985) Ann.Rev.Physiol. 47:545-560). Na+/H+ antiporters exist in various cells of animals and six isoforms (NHE 1 to 6) have been reported (Orlowski, J. and Grinstein, S. (1997) J.Biol.Chem 272:22373-22376).
The first gene cloned for yeast was the gene (sod2) from fission yeast (Schizosaccharomyces pombe) , which was cloned as a gene related to Na+ transport and salt tolerance (Jia, Z. P., McCullough, N., Martel, R., Hemmingsen, S. And young, P. G. (1992) EMBO J. 11:1631-1640). Also, a gene with high identity to this gene has been found from a budding yeast (Saccharomyces cerevisiae), as well as Zygosaccharomyces rouxii (named NHA1 and ZSOD2, respectively) (Prior, C. et al. (1996) FEBS Letter 387:89-93; Watanabe, Y. et al. (1995) Yeast 11:829-838). Two different Na+/H+ antiporter genes (nhaA, nhaB) have been isolated from E.coli (Karpel, R. et al. (1988) J.Biol.Chem. 263:10408-10410; Pinner, E. et al. (1994) J.Biol.Chem. 269:26274-26279), each closely related to Na+ transport and salt tolerance. With respect to plants, activities in algae and such have been examined (Katz, A. et al. (1989) Biochim.Biophys.Acta 983:9-14).
On the other hand, there are only reports on activity in plants for antiporters restricted on vacuolar membranes. To date, Na+/H+ antiporters on the vacuoles have been investigated in connection with salt tolerance in halophytes growing in an environment with high salinity (Matoh, T. et al. (1989) Plant Physiol. 89:180-183; Hassidim, M. et al. (1990) Plant Physiol. 94:1795-1801; Barkla, B. J. et al. (1995) Plant Physiol. 109:549-556), as well as in glycophytes with high salt tolerance, like barley and sugar beet (Hassidim, M. et al. (1990) 94:1795-1801; Blumwald, E. et al. (1987) Plant Physiol. 85:30-33; Garbarino, J. and DuPont, F. M. (1988) Plant Physiol. 86:231-236; Garbarino, J. and DuPont, F. M. (1989) Plant Physiol. 89:1-4; Staal, M. et al. (1991) Physiol.Plant. 82:179-184). The above findings indicate that Na+/H+ antiporters are closely related to salt tolerance of plants. There are several reports on characteristics of Na+/H+ antiporters on the vacuolar membrane. The Km of the antiporter activity for Na+ is about 10 mM similar to that on cytomembrane of mammals (Blumwald, E. et al. (1987) Plant Physiol. 85:30-33; Garbarino, J. and DuPont, F. M. (1988) Plant Physiol. 86:231-236; Orlowski, J. (1993) J.Biol.Chem. 268:16369-16377). Moreover, it is known that amiloride and amiloride derivatives, which are specific inhibitors of Na+ transporters, inhibit the Na+/H+ antiporters on the plant vacuolar membrane and that on the mammalian plasma membrane in a competitive manner (Blumwald, E. et al. (1987) Plant Physiol. 85:30-33; Orlowski, J. (1993) J.Biol.Chem. 268:16369-16377; Tse, C. M. et al. (1993) J.Biol.Chem. 268:11917-11924; Fukuda, A. et al. (1998) Plant Cell Physiol. 39:196-201). These findings suggest the characteristic similarities between Na+/H+ antiporter on the vacuolar membrane of plants and that on mammalian plasma membrane. There are various reports on Na+/H+ antiporter activity of plants as mentioned above, however, in spite of the various trials done, analysis of the substantial part, namely genes as well as proteins thereof, were still left behind (Katz, A. et al. (1989) Biochim.Biophys.Acta 983:9-14; Barkla, B. and Blumwald, E. (1991) Proc.Natl.Acad.Sci.USA 88:11177-11181; Katz, A., Kleyman, T. R., and Pick, U. (1994) Biochemistry 33:2389-2393).
Recently, a gene expected to encode a protein that shares amino acid sequence homology with known Na+/H+ antiporter has been cloned from Arabidopsis; however, the function of this gene remains to be resolved (M. P. Apse et al. (1998) Final Programme and Book of Abstracts “11th International Workshop on Plant Membrane Biology”, Springer; C. P. Darley et al. (1998) Final Programme and Book of Abstracts “11th International Workshop on Plant Membrane Biology”, Springer).
Examples of Na+/H+ antiporter genes isolated from plants are only those isolated from Arabidopsis, a dicotyledon, described above. No isolation of genes from monocotyledoneae, including species such as rice and maize, which are industrially useful crops, have been reported until now.